Method of detection of amino acid sequence and/or identification of peptides and proteins, by use of a new derivatization reagent and synthesis of 5-formyl-benzene-1,3-disulphonic acid as derivatization reagent

ABSTRACT

Present invention refers to a novel and improved method of derivatization and detection of amino acid sequence and/or identification of proteins, peptides by a new derivatization compound. Precisely, the method discloses a novel approach to derivatization of peptides or proteins by compounds comprising two or more sulfonyl groups and analysis of derivatized analytes in negative mode of operation of mass spectrometer. This method allows unambiguous analysis of amino acid sequence of long-chain peptides/proteins. Also, the invention discloses a novel synthesis procedure of 5-formyl-benzene-1,3-disulphonic acid as derivatization compound.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 31, 2015, isnamed 045073-20_SL.txt and is 747 bytes in size.

The subject invention refers to a novel and improved method of detectionof amino acid sequence and/or identification of proteins, peptides.Precisely, the method discloses a novel approach to peptide or proteinderivatization and analysis of resulting analytes in negative mode ofoperation of mass spectrometer with possibility of amino acid sequencingin positive ion mode. Furthermore, the invention also relates tosynthesis of 5-formyl-benzene-1,3-disulphonic acid used asderivatization agent in the foregoing method.

Technical Problem

Protein identification by peptide mass fingerprinting (PMF) using theMS/MS techniques (tandem mass spectrometry) post-source decay (PSD) orcollision-induced dissociation (CID) is based on the comparison ofexperimentally derived data with theoretically calculated peptide massesand fragmentation ions masses in databases (Gevaert et al.Electrophoresis 2001). However, since genome sequence of most organismsis still unraveled, information on particular proteins is not includedin existing databases. In addition, even if relevant protein informationexisted in the databases, different modifications such aspost-translational modifications can hamper identification of a portionor a complete amino acid sequence. Therefore, complete determination ofprimary protein structure requires detection of amino acid sequence withthe minimal use of databases, i.e., de novo peptide and proteinsequencing (H. Steen et al. Mol. Cell. Biol. 2004). The latter is basedon tandem mass spectrometry, MS/MS or PSD experiments. In order tofacilitate interpretation of complex spectra, peptides are chemicallyderivatized by appropriate reagents that, either almost exclusively orin most of the cases, give rise to one series of fragment ions. Massdifference between consecutive signals reveals amino acid sequence.Keough's idea of binding acidic group with N-terminus of peptide hasproved successful. Such derivatized peptide bearing positive andnegative charge at the same time can be depicted by the followingformula: ⁻O₃S—C₆H₅-A₁-A₂-A₃-A₄-A₅-X⁺, where A stands for any amino acid,and X for lysine or arginine (T. Keough et al. Proc. Natl. Acad. Sci.1999, T. Keough et al. Rapid Commun. Mass Spectrom. 2000).

Current procedures of peptide or protein derivatization yield betterresults in comparison with complex analysis of non-derivatized peptides.This refers to derivatization of amino end of peptides with moleculeanalogues with one (Y. H Lee. et al. Proteomics 2004) or two or moresulfonyl groups (M. Cindrić & associates, Patent Application No.P20100044A, HR). Spectra obtained after peptide derivatization withreagent having ortho, para arrangement of sulfonyl groups (with respectto the reactive compound group) demonstrated sufficiently intense ionsignals close to the site of derivatization, whereas distancing from thesite of derivatization the signal would fade away or be lost.Ultimately, this method (M. Cindrić & associates, Patent Application No.P20100044A, HR) demonstrated very poor signal intensity for ions moredistant from the site of derivatization and could not be used in thederivatization and detection of ions several amino acids away from thesite of derivatization. (e.g. distance of 10 and more peptide bonds fromthe site of derivatization). Hence, the method of the foregoingapplication had a limited scope of successful amino acid sequencedetection.

STATE OF THE ART

The most common sulfonyl group-containing reagents used in peptide orprotein derivatization are 2-sulfobenzoic acid (T. Keough et al. Proc.Natl. Acad. Sci. 1999), sulfo-NHS esters (N-hydroxysuccinimide, NHS; W.R. AIlery et al. J. Prot. Research 2007), and 4-sulfophenylisothiocyanate (P. Chen et al. Rapid Commun. Mass Spectrom. 2004), whichrelatively quickly (up to 30 minutes) modify peptide and make itamenable to sequencing. Current literature describessulpho-derivatization reagents based on the following reactive groups:isothiocyano (Y. H. Lee et al. Proteomics 2004), isocyano (P. Conrottoet al. J. Biomol. Techn. 2005), cyclic anhydride (T. Keough et al. Proc.Natl. Acad. Sci. 1999), and N-Hydroxysuccinimide (W. R. AIlery et al. J.Prot Research 2007). Patent application P20100044A, HR describespeptide/protein derivatization by use of derivatization reagent4-formylbenzene-1,3-disulphonic acid having two sulfonyl groups in orthoand para position. Derivatization by use of said compound producessignals whose intensity becomes lower as the distance among detectedions and the site of derivatization increases.

In the state of the art there are several patents/patent applicationsthat reveal different methods of detection of amino acid sequence and/oridentification of proteins, peptides. However, none of the below stateddocuments allows detection of intensive signals of ions in negative modeof operation of mass spectrometer which are distant from the site ofderivatization.

The PCT/US00/00790 patent application describes a procedure based on theuse of the compounds with one or more acidic groups with pKa lower than2 for derivatization of peptide N-terminus. Furthermore, this inventionimplies that derivatized y-ions are used for analysis of fragments bymass spectrometry, which are devoid of a- and b-ions. Althoughderivatives of disulphonic acids are also mentioned as acidic groups,derivatization procedure described in this document is not used forspectra analysis in negative MS/MS mode

Patent document PCT/US01/22815 refers to derivatization oflysine-containing peptides. As in the document mentioned above,derivatization procedure in this document is not used for spectraanalysis in negative MS/MS mode. Furthermore, this document refers toguanidination, i.e., imidolization of lysine, which is not necessarywith the use of the subject invention.

Patent application PCT/US02/16244 describes the use of water-stablereagents for peptide derivatization. The reagents comprise one or moresulfonyl groups bound with activated acidic group via aliphatic oraromatic linkage. Activated acid derivatives described in this patentapplication are acid esters, anhydrides of organic and inorganic acids.The activated acidic moiety particularly mentioned in this patent is NHSester, allowing for all procedure steps to be carried out under aqueousconditions. The four basic steps in the subject invention includeguanidination of lysine. Furthermore, this invention refers to they-ions analysis in positive mode of operation of mass spectrometer.Derivatization procedure in this document is not used for spectraanalysis in negative MS/MS mode.

Patent document PCT/US02/16247 holds priority of the applicationPCT/US02/16244, and, therefore, shares a high level of similarity withthe latter patent. In comparison with the aforementioned patent, it hasbeen added that polypeptides are immobilized on the solid support, atleast in the step “a”. This means that complete derivatization procedureis not carried out in solution, the latter being the case with subjectinvention. In addition, derivatization procedure in the aforementioneddocument is not used to analyse spectra in negative MS/MS mode.

European patent application EP 1561755 describes compounds withdisulfide bond that react with peptide N-terminus followed by disulfidebond cleavage under oxidation or reduction conditions resulting in theformation of sulfonic acid derivatives. Functional group of disulfidecompound that reacts with N-terminus was chosen among carboxyl group,isothiocyanate, succinimidyl oxycarbonyl groups, p-nitrophenoloxycarbonyl groups, pentafluorophenyloxy carbonyl groups, andtetrafluorosulpho phenyloxycarbonyl groups. Subject invention is usedfor detection of amino acid sequence by y-ions analysis in positive modeof operation of mass spectrometer. In this invention, guanidination oflysine is also necessary. In addition, derivatization procedure in theaforementioned document is not used for spectra analysis in negativeMS/MS mode.

Patent application PCT/SE2005/000187 improves previous methods in such away that removal of unmodified portions of peptides from the solution byion exchange precedes the analysis of fragments in positive mode ofoperation of mass spectrometer. This procedure is used after anychemically-aided peptide derivatization.

Accordingly, conventional procedure of peptide derivatization in thestate of the art was carried out by introduction of sulfonyl groups toN-terminus. N-terminus derivatized in this manner becomes negativelycharged. Positively charged C-terminus is a counterbalance to negativelycharged N-terminus resulting in the formation of the so caIledzwitterion, i.e., charge of derivatized protein or peptide equals zero.In further ionization procedure in mass spectrometer used in the stateof the art, proton was added to peptide/protein reducing the energynecessary for peptide bonds cleavage, which produced mostly b- andy-ions. Since b-ions would be neutral due to negative charge atN-terminus, only positive y-ions in positive mode of operation of massspectrometer were analysed in the state of the art.

Patent application WO 00/20870 describes a method of multiple cleavagesusing a variety of reagents. After polypeptide cleavage, the isolationprocess is described in detail. The method is designed to isolateparticular peptides from the rest of the solution by attaching cleavedpeptides to the solid phase through chromatography. The method does notimprove the fragmentation process of peptides in mass spectrometer.Patent application WO 96/02003 describes use of newly synthesizedalkoxy-thiocarbonyl-imidazole with smaIler peptides cleavage. The newlysynthesized reagent is an alkoxy thiourea derivative and binds topeptide N-terminus. The mechanism of its activity includes cleavage inthe solution and use of acid, which cleaves off terminal amino acid inthe form of thiazolinone derivative which cannot rearrange to athiohydantoin. Thiazolinone can have fluorescent marking and analyzed bymass spectrometer. The method is adjusted to the operation in thesolution and there are no data confirming improvement of fragmentationin mass spectrometer.

Patent application EP 1617224 describes ionization and fragmentationimprovement in mass spectrometry by derivatization of peptide C-terminusby 2-methoxy-4,5-dihydro-1H-imidazole. Proposed derivatization andfragmentation mechanism by use of mass spectrometry has no similaritywith sulfonyl derivatization reagents operation mechanism. The basicityof derivatized lysine (Lys-4,5-dihydro-1H-imidazole fragment) is indirect correlation with ion intensity in mass spectrometer. Secondaryamines bound to lysine by derivatization with2-methoxy-4,5-dihydro-1H-imidazole are one of the strongest non-ionicbases, i.e., the mechanism of their activity is connected with strongproton binding with peptide. Completely opposed to said mechanism is themechanism of activity of sulfonyl groups. Sulfonyl groups releaseprotons and in this manner they increase peptide acidity and facilitatefragmentation in mass spectrometer.

Patent application P20100044A, HR describes a process of using4-formyl-benzene-1,3-disulphonic acid (two sulfonyl groups in ortho andpara position with respect to the aldehyde reactive group) for detectionof amino acid sequences. The arrangement of sulfonyl groups situated inortho and para position in benzene hinders donating of labile proton todistant amino acid amides, reducing the possibility of detection ofamino acids distant from the site of derivatization. Consequently,incomplete amino acid sequence and/or protein or peptide detectionresults are obtained.

DISCLOSURE OF THE INVENTION

Based on the knowledge comprised in the state of the art that refers tomethods of detection of amino acid sequence and/or identification ofproteins, peptides, in particular the patent application HR P20100044A,it has become clear that the methods known so far could not give goodresults of protein or peptide detection wherein it was necessary todetect signals of ions that are distant from the site of derivatization.As the main objective of derivatization reagent activity is to achieve aseries of most intensive signals and, thus, determine unambiguously theamino acid sequence of the entire peptide, the compound of generalformula I, i.e., two sulfonyl groups in optimum position (meta, meta) insaid compound enable the determination of intensive signals which couldnot be revealed with different sulfonyl group arrangement (e.g. ortho,para). Determination of the entire sequence of peptide or of its majorportion increases unambiguousness of the result obtained and facilitatesidentification of proteins with increased correctness and accuracy ofmeasurement.

Considering that derivatization reagents containing two or more sulfonylgroups which were commercially available did not provide good results,non-commercially available compounds which are not usually applied orare rarely applied in various researches were synthesized.

Compounds of general formula I were obtained by different synthesisprocedures

where R is a reactive group selected from aldehyde-, keto-,isothiocyanate-, isocyanate-group, NHS ester, anhydride or activatedcarboxylic acid group.

In the foregoing compounds group, sulfonyl groups are in meta, metaposition with respect to the reactive compound group. Upon examinationof derivatization compounds of general formula I in the method ofdetection of amino acid sequence and/or identification of proteins,peptides in negative mode of operation, it was unexpectedly establishedthat such synthesized compounds of general formula I allow for moreprecise (detailed) detection of amino acid sequence and/oridentification of proteins, peptides in comparison with the compounds innegative mode of operation used so far.

Namely, by applying obtained compounds of general formula I in thederivatization procedure, it was established that by use of compound ofgeneral formula I as derivatization reagent in negative mode ofoperation of mass spectrometer, the signal intensity of ions distantfrom the site of derivatization is unexpectedly multiply increased incomparison with the earlier use of 4-formyl-benzene-1,3-disulphonic acidor another compound containing two sulfonyl groups in ortho and paraposition with respect to the reactive group. Ions distance from the siteof derivatization where such unexpected effect of the compound ofgeneral formula I is noticeable depends on detected protein, peptide oramino acid sequence. Current results have shown that the unexpectedeffects of this invention generally appear at the distance at which ionintensity commences decreasing with the use of compound containing twosulfonyl groups in ortho and para position with respect to the reactivegroup. That value dependent upon amino acid sequence appears in ions ofamino acids distant from the site of derivatization (distance from thesite of derivatization where increase in ion intensity commences to benoticeable can vary from 5 to 10 amino acids).

Use of the compound of general formula I in the method of detection ofamino acid sequence in negative mode of operation enables unambiguousanalysis of the signal of ions in negative mode of operation of massspectrometer which are distant from the site of derivatization.Considering that use of derivatization compounds used so far in negativemode of operation enabled no analysis of the signals of ions distantfrom the site of derivatization, change of the compounds of subjectinvention in the foregoing method provides more detailed and accurateresults in comparison with derivatization compounds used so far in saiddetection method.

Therefore, subject invention reveals an improved method of detection ofamino acid sequence of peptides/proteins, i.e., method of identificationof peptides/proteins comprising the following steps:

-   -   derivatization of peptides and/or proteins at N-terminus by        compound of general formula I comprising R reactive group that        binds with amino group of N-terminus;    -   analysis of one or more derivatized analytes by acquisition of        spectra of derivatized negative ions in negative mode of        operation of mass spectrometers;    -   interpretation of obtained fragmentation pattern as to detect        amino acid sequence, i.e., to identify the aforementioned        analyte.

The subject step of derivatization of peptides and/or proteins givesrise to exclusively one series of fragmented ions. Precisely, this stepproduces singly charged derivatized negative ions. During derivatizationin the subject invention with the compound comprising two sulfonylgroups, one sulfonyl group neutralizes positive ion charge, whereas theother sulfonyl group gives negative charge to derivatized ion. If thesulfonyl groups are situated at equidistant meta, meta distance from thesite of derivatization, proton donation necessary for cleavage of amidebonds and production of signals is more pronounced. This new andunexpected effect allows detection of obtained derivatized negative ionsin negative mode of operation of mass spectrometer, especially if ionsare several amide bonds away from the site of derivatization incomparison with any known means of derivatization.

Unlike the aforementioned invention, current procedures of peptide orprotein derivatization did yield better results in comparison withcomplex analysis of non-derivatized peptides. This refers toderivatization of amino end of peptides with molecule analogues with one(Y. H Lee. et al. Proteomics 2004) or two or more sulfonyl groups (M.Cindrić & associates, Patent Application No. P20100044A, HR). However,spectra obtained after peptide derivatization with reagent having ortho,para arrangement of sulfonyl groups demonstrated limitations in theanalysis of distant amino acids. Since ortho group is in directproximity of the site of derivatization the majority of protons donatedby ortho-sulfonyl group in mass spectrometer is consumed for cleavingoff active benzo-disulfonyl group of reagents (intensive signal visiblein negative MS/MS spectrum at m/z 249). Cleaving off activebenzo-disulfonyl group reduces further reagent activity in massspectrometer, which manifests by reduction of mobile proton range.Distancing the sulfonyl group from the site of derivatization andplacing sulfonyl groups into equidistant position as in the case ofmeta, meta arrangement of sulfonyl groups prevents completelyderivatization reagent to be cleaved off during analysis, increasingthereby mobile proton range and intensity of the signal of cleaved offpeptide bonds which are distant from the site of derivatization (signalin negative MS/MS spectrum at m/z 249 is absent). The range of mobileproton is connected with the nature of analyzed peptides, but in thedirect comparison between ortho, para sulfonyl groups and meta, metasulfonyl groups, meta, meta arrangement has always resulted in broadermobile proton range and, thus, also in more intensive signals in totalstarting from the site of derivatization to C-terminus.

Therefore, unlike peptide derivatization by compounds comprising twosulfonyl groups with no equidistant meta, meta distance among sulfonylgroups and the reactive group (patent application P20100044A, HR), inthe compounds of general formula I, where sulfonyl groups are in meta,meta position with respect to the reactive group, interference ofadjacent molecular groups is reduced and easier proton donation isallowed. The advantage of the subject method is that the signal detectedin MS/MS scan of derivatized negative ions predominates over the signalsof positive y ions obtained by derivatization by the compoundscomprising one sulfonyl group for at least 5-fold in an absolute amountmeasured for an equal amount of analyte applied onto MALDI plate.Furthermore, the advantage of the subject method over the analysis ofpeptide ions comprising two sulfonyl groups which are not equidistantwith respect to the reactive group in the molecule is an increasedproton range in negative mode of operation measured for an equal amountof analyte applied onto MALDI plate.

Finally, use of the compound of general formula I increases reactivegroup reactivity, as meta, meta sulfonyl groups are two chemical bondsaway from the site of aldehyde binding with amine whereby mobile protonrange is improved, facilitating peptide analysis with increase of signalintensity of amino acid ions distant from the site of derivatization.

Furthermore, upon synthesizing of the compounds of general formula I, aso far unknown synthesis procedure of 5-formylbenzene-1,3-disulphonicacid was developed. The new synthesis procedure of5-formylbenzene-1,3-disulphonic acid comprises the following steps:

-   -   Conversion of 3,5-dihydroxybenzaldehyde into        O-benzaldehyde-3,5-bis(N,N-dimethylthiocarbamate) by use of        relevant N,N-dialkylcarbamoylchloride with organic or inorganic        bases in suitable organic solvent;    -   Rearrangement of        O-benzaldehyde-3,5-bis(N,N-dimethylthiocarbamate) to obtain        S-benzaldehyde-3,5-bis(N,N-dimethylthiocarbamate); rearrangement        reaction can be performed thermally without solvent or with        relevant high-boiling solvent at 150-280° C. or catalytically by        use of palladium complex or Lewis acid);    -   Conversion of S-benzaldehyde-3,5-bis(N,N-dimethylthiocarbamate)        into S-benzylalcohol-3,5-bis(N,N-dimethylthiocarbamate) by        catalytic hydrogenation or reduction with complex metal        hydrides, and then into        S-benzylacetate-3,5-bis(N,N-dimethylthiocarbamate) by use of        acetic acid chloride or anhydride;    -   Oxidation of S-benzylacetate-3,5-bis(N,N-dimethylthiocarbamate)        into benzylalcohol-3,5-disulphonic acid with suitable organic or        inorganic oxidant;    -   Oxidation of benzylalcohol-3,5-disulphonic acid into        5-formylbenzyl-1,3-disulphonic acid by use of pyridinium        chlorochromate.

The reaction scheme of the new synthesis procedure of5-formyl-benzene-1,3-disulphonic acid is as follows:

In said synthesis the reactions of oxidation and reduction have beenperformed on the target substituent successfully, despite bothfunctional groups being susceptible to both oxidations and reductions.Introduction of additional reactions: aldehyde reduction, acetylprotection, deprotection and again oxidation enabled to carry outtargeted desired reactions of oxidation and reduction and to obtain as aresult compound 5-formyl-benzene-1,3-disulphonic acid.

The term “derivatization compound” in the subject invention refers tothe compound of general formula I.

The term “reactive group” in the subject invention refers to anyfunctional group known to the person skiIled in the relevant art toreact with amino group.

The term “analyte” in the subject invention refers to any portion(segment) of peptide and/or protein produced in the derivatization step,analysed in mass spectrometer.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 represents the route of synthesis of5-formyl-benzene-1,3-disulphonic acid (synthesis is described in“Detailed description of the invention”);

FIG. 2 represents cleavage mechanism of analytes in mass spectrometer;

FIG. 3 represents MS/MS mass spectra for equal amounts of analyzedanalytes of tryptic digests of trypsin autolysis (1 ng) of: (3A)LeuGlyGluHisAsnIleAspValLeuGluGlyAsnGluGlnPheIleAsnAlaAlaLys peptide SEQID NO: 1) derivatized by derivatization compound4-formyl-benzene-1,3-disulphonic acid with two sulfonyl groups in orthoand para position acquired in the negative mode of operation of massspectrometer and (3B)LeuGlyGluHisAsnIleAspValLeuGluGlyAsnGluGlnPheIleAsnAlaAlaLys peptide SEQID NO: 1) derivatized with derivatization compound5-formyl-benzene-1,3-disulphonic acid with two sulfonyl groups in metaand meta position acquired in negative mode of operation of massspectrometer.

DETAILED DESCRIPTION OF THE INVENTION Analysis of Peptides/ProteinsUsing the Compound of General Formula I

The method presented by the subject invention enables detection ofcomplete amino acid sequence of proteins, i.e., peptides. In proteinidentification, protein cleavage with known chemical or enzymaticproteolytic procedures precedes the method of the subject invention.Thus, proteins can be cleaved for example with chemical compounds suchas ninhydrin, cyanogen bromide or by simple degradation using acidhydrolysis. Furthermore, proteins can be cleaved with enzymes such astrypsin, chymotrypsin, thermolysin, Lys-C, Glu-C, Arg-C, etc.

The procedure of protein proteolysis, involving in the majority of casespeptides up to 5000 Da, with the possibility of derivatization of largerpeptides as well, is followed by peptide mass determination by massspectrometry and thereafter peptide derivatization procedure.Derivatization compound of general formula I is added to aqueoussolution of peptides/proteins to be derivatized. Derivatization compoundbinds to N-terminus of peptide via reactive group giving it two sulfonylgroups and increasing its mass. Therefore, during the mass spectrometry,it is necessary to add thereto the m/z value of the relevantderivatization compound comprising two sulfonyl groups to the peptidemass (in MS/MS positive mode of operation the reagent is detached duringanalyses yielding products; y-ions that can reveal amino acid sequence,whereas in MS/MS negative mode of operation the reagent is not detachedfrom N-terminus, but the formation of products; derivatized b-ionsrevealing amino acid sequence is still increased). Therefore, N-terminusis doubly negatively charged, whereas C-terminus or some basic sidebranch of peptide and/or protein is positively charged. When sulfonylgroups donate labile protons to amide groups, peptide and/or proteindissociates into amino acid integral portions. Resulting positive ionsdo not differ in mass from the positive ions of non-derivatizedanalogue, since sulfonyl groups during proton donation to amide bond inthe peptide cleave off either a portion of ions from N-terminus ofpeptide chain or derivatization compound itself, thus producinggradually shorter peptide/protein ions. The same mechanisms in negativemode of operation yields the final reaction products, i.e., negativelycharged derivatized ions comprising two sulfonyl groups at N-terminus,which increase the mass of precursor ions and product ions for the massvalues of derivatization reagent (FIG. 2). However, regardless of theaddition of derivatization group, the differences between detected ionsin positive or negative mode of operation of mass spectrometry areequal, which ultimately enables determination of amino acid sequence ortheir modifications. The arrangement of sulfonyl groups with respect tothe site where aldehyde of derivatization compound binds with peptideamine is equidistant (meta, meta), i.e., both groups are equally distantfrom the binding site, but they are not in direct proximity of reactivegroup. Such arrangement of sulfonyl groups with respect to the otherdisulfonyl analogues allows easier proton donation to amide groups onthe peptide.

Derivatized negative ions can be analysed in different massspectrometers. Thus, the following mass spectrometers are used:time-of-flight (TOF), tandem mass analyzers (MS/MS, QQQ, MS/Q, Q/TOF),quadropole (Q), ion trap (IT) and similar devices. Moreover, experimentshave shown that the use of MALDI ion source in the subject inventiongives rise to exclusively y fragments of positive ions and derivatizednegative b-ions. Signals obtained by analysis of these derivatizednegative b-ions produced in the subject procedure are extremelyintensive, whereas noise in comparison with standard methods ofdetection of amino acid sequence or identification of peptide/protein isup to 10-fold lower. Thus, the overall increase of signal intensityobtained by the subject invention is up to 15-fold higher than thesignal intensities obtained by the methods in the state of the art,except in the case of derivatization by other derivatization compoundswhere sulfonyl groups are in ortho, para position with respect to thereactive group.

In the particular example compared results are obtained byderivatization with the use of 4-formyl-benzene-1,3-disulphonic acid(compound of patent application P20100044A) and5-formyl-benzene-1,3-disulphonic acid. In comparison with derivatizationwith the use of 4-formyl-benzene-1,3-disulphonic acid the improvement ofsignal intensity by amino acids which are 10 and more amino acids awayfrom the site of derivatization is up to 10-fold higher with respect tothe noise after peptide derivatization with5-formyl-benzene-1,3-disulphonic acid (FIG. 3A and FIG. 3B and Table 1).Improvement of mobile proton range is allowed by different arrangementof sulfonyl groups (meta, meta in 5-formyl-benzene-1,3-disulphonic acidwith respect to ortho, para in 4-formyl-benzene-1,3-disulphonic acid).Ortho sulfonyl group donates the majority of its protons to the nearestamine produced after derivatization of N-terminus, which is visible inspectra of peptide cleavage in negative mode of operation of massspectrometer where measured signal at m/z 249 is often the mostintensive in the spectrum (signal produced after reagent cleavage at thesite of derivatization). Such pronounced derivatization reagent cleavingoff is absent when sulfonyl groups are in meta, meta position. Moreover,meta, meta sulfonyl groups do not hinder derivatization of N-terminus asnoticed when sulfonyl groups are in direct proximity of benzaldehydegroup (ortho position). Due to said reasons ortho sulfonyl group hindersderivatization and uses the majority of its protons to cleave off thenearest nitrogen reducing the activity range of mobile proton towardsdistant amino acids. The comparison was carried out under the sameconditions in mass spectrometer and with the same amount ofderivatization peptides (1 ng of tryptic digest of trypsin autolysis).However, by using mass spectrometers that cleave peptides/proteins in adifferent manner, it is possible to obtain with the subject inventionalso other fragments of negative and positive ions, maintaining thebasic component of amino acid sequence determination based on thedifference between masses of signal sequence.

Direct comparison of the compound used in the state of the art(4-formylbenzene-1,3-disulphonic acid) and the newly synthesizedcompound (5-formylbenzene-1,3-disulphonic acid) reveals increase ofsignal intensity of b-ions of amino acids, and extended range ofderivatization reagent. This increase is most contributed by meta, metaarrangement of sulfonyl groups which do not sterically hinder thederivatization procedure, and do not enter into interaction in massspectrometer with the nearest amino or amide protons.

TABLE 1 Comparison of signal intensity of peptide b-ions 10 or moreamino acids away from the site of derivatization ofLeuGlyGluHisAsnIleAspValLeuGluGlyAsnGluGlnPheIleAsnAlaAlaLys peptide(SEQ ID NO: 1) after derivatization with 4-formyl-benzene-1,3-disulphonic acid (ortho, para) and with 5-formyl-benzene-1,3-disulphonicacid (meta, meta). Signals of b-ions less than 10 amino acids away werenot taken into calculation considering that in that field calculated S/Nis the same for both derivatization reagents. Distance from the Measuredsignal-to- Fragment Amino site of noise ratio (S/N) m/z acidderivatization (ortho, para)/(meta, meta) 1368 Glu 10 98, 5/533, 6 1425Gly 11 20, 1/86, 8  1539 Asn 12 32, 0/140, 2 1668 Glu 13 23, 4/150, 71796 Gln 14  8, 4/107, 8 1943 Phe 15 8, 0/53, 9 2056 Ile 16  8, 9/101, 72170 Asn 17 8, 7/51, 2 2241 Ala 18 0, 0/15, 5

Reactive group of derivatization compound can be any group reacting withamino group. It is preferable that reactive group of the compound usedin derivatization step of the subject invention is selected from thegroup comprising aldehyde, keto-, isothiocyanate-, isocyanate-group, NHSester, anhydride or activated carboxylic acid group.

It is most preferred that, in the subject invention, reactive group ofderivatization compound is aldehyde group. In the cases where in thesubject invention compound with aldehyde reactive group at ph 1-5 isused, aldehyde group selectively reacts with N-terminus of peptide orprotein, without binding to other amino groups in protein or peptidechain. By using this approach dual derivatization of tryptic peptides isavoided, since derivatization reaction performed according to thesubject invention selectively derivatized N-terminus without the need ofadding protective group at lysine. Since in this case lysineguanidination is redundant, the method of detection of amino acidsequence, i.e., peptide/protein identification is additionallysimplified. Also, since guanidination, which causes significantquantitative losses during sample handling, is not needed, signalintensity in the analysis of derivatized negative ions in negative modeof operation of mass spectrometry is increased. It is most preferredthat compound with aldehyde group in derivatization step is used atapproximately pH 4. Furthermore, it is preferable that during the use ofderivatization compound with aldehyde reactive group, NaCNBH4 is alsoadded to aqueous solution as to reduce imino-group of the resultingSchiff base.

Moreover, it is most preferred to use 5-formyl-benzene-1,3-disulphonicacid as compound in derivatization step.

One of the Embodiments of the Invention Example 1 Use of5-formylbenzene-1,3-disulphonic acid as a Derivatization Compound

In this example the method of peptide derivatization of the subjectinvention by chemical reaction in two stages was used: first stageincludes condensation of aldehyde and primary amine with production ofSchiff base, and the second stage includes reduction of imines of Schiffbase into amines. The reagents used included:5-formyl-benzene-1,3-disulphonic acid (synthesized at “Rudjer Boskovic”Institute), NaCNBH4 (Merck, Darmstadt, Germany), and peptides obtainedby trypsin autolysis (Merck, Darmstadt, Germany), CHCA matrix(α-cyano-4-hydroxycinnamic acid, Sigma Aldrich, St. Louis, Wis., USA).

Table 2 presents trypsin peptide and its ion detected by massspectrometry, which are produced during trypsin autolysis prior toderivatization by 5-formyl-benzene-1,3-disulphonic acid.

TABLE 2  Peptide produced by trypsin autolysis withtheoretical calculation of masses of corresponding ions. FRAGMENT m + Hsequence (SEQ ID NO: 1) T3 2211.1000 LeuGlyGluHisAsnIleAspValLeuGluGlyAsnGluGlnPheIleAsnAlaAlaLys

Upon purification of 1 μg fragments derived from trypsin autolysis usingZipTip technique, peptide mixture was evaporated using SpeedVac(Eppendorf, Germany) concentrator followed by addition of 1 mg5-formyl-benzene-1,3-disulphonic acid and 4 mg NaCNBH₄ dissolved in 100μl phosphate buffer pH 4.0 to dried concentrate. The solution was storedin the fridge at temperature of 4-8° C. for 12 hours to react.Subsequently, 10 μl of the solution was purified again using ZipTip,dried in SpeedVac concentrator and dissolved in 5 μl CHCA matrix with 5mg/ml concentration. 1 μl of this solution was applied onto MALDI metalplate and analysed by mass spectrometer.

Derivatization procedure of peptides (SEQ ID NO: 1) obtained by proteincleavage by trypsin at N-terminus is illustrated by the followingreaction:

The example of instrumental MS/MS fragmentation of analytes originallyderived from trypsin autolysis shows significant improvement ofstructural analysis of produced analytes, which increases accuracy ofamino acid sequence detection (so caIled de novo sequencing of unknownpeptides).

For the purpose of evaluation of method success, MS/MS spectra wereacquired on MALDI-TOF/TOF instrument before and after derivatization. Anexample presented in FIG. 3 gives a comparison of MS/MS spectra ofnegative b-ions of ion derivatized by 4-formylbenzene-1,3-disulphonicacid of lysine fragmentLeuGlyGluHisAsnIleAspValLeuGluGlyAsnGluGlnPheIleAsnAlaAlaLys (SEQ IDNO: 1) (FIG. 3A) and MS/MS spectra of negative b-ions afterderivatization by 5-formyl-benzene-1,3-disulphonic acid (FIG. 3B). It isimportant to point out that MS/MS spectra of negative derivatized ionsare acquired on the same sample, i.e., on the same ions obtained afterderivatization, and that the amount of used analyte was the same as theconditions of acquisition of both derivatized peptides.

Produced negative b ions differ in mass for m/z 247,945 in comparisonwith underivatized fragments. During MS/MS analysis of negativelycharged ions of derivatized fragment, it is possible to detect only bions that kept sulfonyl groups, which requires that an increment of m/z247,945 is added (FIGS. 2A and 2B). Extremely intensive signal producedby cleaving off derivatization reagent m/z 249 (247,945+1H⁺) is absentin the case of use of 5-formyl-benzene-1,3-disulphonic acid which provedhigher mobile proton affinity for distant amino acids (FIG. 3B).Extremely intensive signal m/z 249 indicates consumption of largeportion of protons situated in ortho position in sulfonyl group oncleavage of the nearest amino group (site of derivatization, i.e.,derivatization reagent).

Comparison of spectra in FIGS. 3A and 3B shows significant increase inmeasured ion signals in spectrum in FIG. 3B in comparison withequivalent signals in FIG. 3A (signals ten and more amide bonds awayfrom the site of derivatization), which proves that the subject methodincreases success in detection of amino acid sequence of peptides.

If up to 10-fold lower noise detected in analysis of derivatized ionswith 5-formylbenzene-1,3-disulphonic acid is added, the overall increasein signal-to-noise ratio (S/N) after derivatization is 15-fold for MS/MSnegative ions. If ions of amino acids presented in FIGS. 3A and 3B arecompared, significant difference is noticeable after the tenth signal inamino acid sequence (m/z 1368) in favour of signal increase of peptideions derivatized with 5-formyl-benzene-1,3-disulphonic acid (Table 1 andFIG. 3B). Similar experiments were carried out on all ions consisting of10 or more amino acids presented in Table 1 before and afterderivatization, and obtained results do not differ from the results setout in this example.

Method described in the subject patent application is technically rapid,cost effective and reliable, and could be of high value in proteomicsanalyses of various samples, especially the biological ones. In fact,only in humans the size of the whole proteome is estimated to severalmillion protein species, and databases currently provide information oncompletely determined amino acid sequences for approximately 1.5 millionproteins. The described method could, thus, facilitate simpledetermination of amino acid sequences of human proteins unidentified sofar with significant medical implications (e.g. biomarker discovery). Itwould also be as simple to identify proteins for different biologicalspecies for which publicly accessible protein databases, such as NCBIand UNIPROT, contain no exact information on amino acid sequences.

Subject method reduces the time of sub-structural analyses of proteinsand peptides by mass spectrometry (MS/MS analysis) and increasesaccuracy when searching protein databases.

Detailed Description of 5-formyl-benzene-1,3-disulphonic acid Synthesis

The numbers of synthesized intermediates and final product correspond tothe numbers in FIG. 1.

1. O-benzaldehyde-3,5-bis(N,N-dimethylthiocarbamate) (2)

To the solution of 3,5-dihydroxybenzaldehyde (630 mg, 4.5 mmol,Sigma-Aldrich, St. Louis, Wis., USA) 3.0 g (22 mmol) K₂CO₃ (Kemika,Zagreb, Croatia) and 1.25 g (10 mmol) N,N-dimethylcarbamoylchloride(Sigma-Aldrich, St. Louis, Wis., USA) were added in 50 ml dryacetonitrile. Reaction mixture was mixed for 24 hours at roomtemperature, 20 ml water and 0.5 g KOH were added and mixed another 30minutes. The majority of acetonitrile was evaporated and water solutionextracted with use of dichlormethane (30+20 ml). Organic extracts werewashed with saturated NaCl solution, dried and evaporated.Pre-crystallization from methanol produced 980 mg (70%) of pure product.

1H NMR (CDCl₃): 3.37 (s, 6H); 3.46 (s, 6H); 7.14 (t, J=2.3 Hz, 1H); 7.49(d, J=2.3 Hz, 2H); 9.98 (s, 1H) ppm.

13C NMR (CDCl₃): 38.90; 43.39; 121.33; 124.30; 137.53; 154.55; 186.77;190.08.

2. S-benzaldehyde-3,5-bis(N,N-dimethylthiocarbamate) (3)

Carbamate 2 (350 mg, mmol) was heated in 2 ml diphenyl ether(Sigma-Aldrich, St. Louis, Wis., USA) at 230° C. for 2 hours. With useof chromatography on silica gel column (Sigma-Aldrich, St. Louis, Wis.,USA) in addition to hexane-dichlormethane-ethylacetate, 315 mg (90%) ofpure product was obtained.

1H NMR (CDCl₃): 3.04 (brs, 6H); 3.08 (brs, 12H); 7.88 (t, J=1.6 Hz, 1H);8.00 (d, J=1.6 Hz, 2H); 9.99 (s, 1H) ppm.

13C NMR (CDCl₃): 37.08; 131.16; 136.97; 137.25; 147.39; 165.53; 190.55ppm.

3. S-benzylalcohol-3,5-bis(N,N-dimethylthiocarbamate) (4)

Thylthiocarbamate 3 (315 mg) was dissolved in 40 ml ethanol and 50 mgNaBH₄ (Sigma-Aldrich, Buchs, Switzerland) was added. The solution wasmixed for 1 hour at room temperature, hydride residue was destroyed withammonium chloride solution (Kemika, Zagreb, Croatia), ethanol wasevaporated, and the product extracted with use of dichloromethane. Pureproduct in the amount of 320 mg (100%) was obtained.

4. S-benzylacetate-3,5-bis(N,N-dimethylthiocarbamate) (5)

To the solution of compound 4 (320 mg) 1.0 ml triethylamine(Sigma-Aldrich, St. Louis, Wis., USA) and 0.9 ml acetic acid anhydride(Kemika, Zagreb, Croatia) were added in 30 ml dichloromethane. After 3hours anhydride residue was destroyed with addition of methanol.Reaction solution was washed with 2% HCl solution, dried and evaporated.Acetate 5 in the amount of 330 mg (91%) was obtained.

1H NMR (CDCl₃): 2.09 (s, 3H); 3.05 (brs, 12H); 7.51 (d, J=1.2 Hz, 2H);7.60 (t, J=1.2 Hz, 1H) ppm.

13C NMR (CDCl₃): 20.97; 36.98; 65.26; 129.77; 135.83; 137.15; 141.87;166.15; 170.69 ppm.

5. Benzylalcohol-3,5-disulphonic acid (6)

To the cooled mixture of formic acid (15 g, Kemika, Zagreb, Croatia),water (1.8 g) and hydrogen peroxide (30%, 1.5 g, Kemika, Zagreb,Croatia) 125 mg acetate 5 was added. Reaction mixture was mixed for 20hours at room temperature and evaporated to dryness. The raw product waspassed through a column of Amberlite IR-120 (H⁺), (Rohm and Haas,Philadelphia, Pa., USA); the water solution was evaporated to dryness,and the residue was dissolved in 20 ml methanol and heated to boilingpoint for 30 minutes. Evaporation of the solution produced 90 mg (95%)of product 6.

1H NMR (CD₃OD): 4.68 (s, 2H); 7.91 (s, 2H); 8.21 (s, 1H) ppm.

13C NMR (CD₃OD): 62.94; 122.09; 125.53; 142.91; 145.08 ppm.

No alternative oxidation route was found in the literature, and there isthe possibility that other peroxyacids in aqueous acid conditions areable to oxidize the resulting compound in the same manner.

6. Benzaldehyde-3,5-disulphonic acid (7)

Alcohol 6 (100 mg, 0.37 mmol) was dissolved in 30 ml dry acetonitrile.Pyridinium chlorochromate (Prepared according to: E. J. Corey and W.Suggs, Tetrahedron Lett. 1975, 16, 2647-2650) in the amount of 90 mg(0.41 mmol) was added to the solution and mixed for 2 hours at roomtemperature. The solvent was evaporated and 50 ml methanol and 3 mlammonia (25%) were added. After 3 hours the residue was filtered, thesolvent was evaporated, and the remaining was passed through a column ofAmberlite IR-120 (H⁺). Disulphonic acid 7 in the amount of 80 mg (80%)was obtained.

There are many other, more or less alternative reagents for whichauthors claim that they oxidise alcohol into aldehyde.

1H NMR (D₂O): 8.36 (s, 3H); 9.96 (s, 1H) ppm.

13C NMR (D₂O): 126.65; 128.25; 129.05; 137.07; 193.86 ppm.

Shown examples of the methods of derivatization and synthesis of5-formyl-benzene-1,3-disulphonic acid present only some of theembodiments of the subject invention and have no limitation on the scopeof patent protection of the subject invention.

The invention claimed is:
 1. A method of detection of amino acidsequence and identification of peptides and/or proteins, comprising thefollowing steps: selective derivatization of peptides and/or proteins atN-terminus having unprotected epsilon-amine of lysine, wherein theselective derivatization is carried out at pH 1-5 with addition ofsodium cyanoborohydride (NaBH₄CN) using 5-formylbenzene-1,3-disulphonicacid, wherein the selective derivatization comprises reacting of thealdehyde group with an amino group at the N-terminus of the peptide orprotein to form derivatized analytes in a form of a secondary amine;analysis of one or more of the derivatized analytes by acquisition ofspectra of derivatized negative ions acquired in the MS/MS negative modeof operation of a mass spectrometer; and interpretation of an obtainedfragmentation pattern to detect amino acid sequence and to identify theaforementioned analyte.
 2. The method according to claim 1, wherein inthe analysis step, the spectra of derivatized negative b-ions isacquired in the MS/MS negative mode of operation of the massspectrometer.